Once harvested, in order to remain viable and retain their undifferentiated state, stem cells must be preserved prior to use for medical or research purposes.
It is well known that stem cells can be stored indefinitely if stored at the temperature of liquid nitrogen (−196° C.). However, it is well documented that the process of freezing may cause irreparable damage to the cells and as such, various cryopreservative agents can be added to the cell suspension prior to the freezing process. US2005/0106554 discloses methods and compositions for the cryopreservation of pluripotent cells, in particular human embryonic stem cells. The methods disclosed in US2005/0106554 are shown to exhibit an increase in cell viability and a decrease in cell differentiation when compared with conventional methods. The cryopreservation method disclosed in US2005/0106554 comprises encapsulation of the cells between two layers of a solid support matrix, adding a cryopreservative to the matrix-cell-matrix composition, and cooling said composition to a temperature sufficient to cryopreserve the cells. US2005/0106554 discloses a carbohydrate-based medium, preferably trehalose, as a suitable cryopreservative. WO2005/118785 discloses methods for the cryopreservation of stem cells, wherein said method includes performing ice nucleation on a cell suspension prior to the reduction of temperature sufficient to allow long term storage of the stem cells. WO2005/118785 further discloses that the cell suspension may or may not contain any exogenous biological cryoprotectant, such as serum.
A number of non-cryogenic methods of stem cell preservation are also known. In such methods, stem cells are generally stored as a cell dispersion in an aqueous solution containing tissue cell culture growth media. Often a preservative compound is included in such aqueous solutions so as to reduce the rate at which cell viability decreases. In a typical example, U.S. Pat. No. 5,912,174 discloses a method of storing a population of mammalian cells capable of duplication and differentiation by suspending said population of cells in an aqueous mixture containing gelatin. Preferred mixtures contain standard tissue cell culture growth media such as RMPI or Eagle's media. Optionally, cell-specific growth factor may also be added to preserve cell viability and, for optimum storage life, the storage temperature should be maintained between 0 and 4° C. Further, EP-A-1057405 discloses the use of an aqueous storage liquid comprising polyphenol and a storage liquid selected from Euro-Collin's solution, UW solution, serum and antibiotic solution for the in vitro freeze-free preservation of, inter alia, a stem cell, tissue or organ for transplantation. The polyphenols disclosed in EP-A-1057405 include catechins such as epigallocatechin, tannic acid, proanto-dianisidine, resorcinol, hydroquinone, pyrogallol, phloroglucinol, eugenol and quercetin. There is no suggestion in EP-A-1057405 to the effect that any of the disclosed compounds may preserve the viability of undifferentiated cells. EP1627565 discloses a medium for storing biological samples in a refrigerated, frozen or vitrified state, comprising a balanced salt solution and 4-thioderivative of flavon-3-ol. WO 2006/019366 discloses a culture system developed for the culturing of human embryonic stem cells. The culture condition includes culture of the cells in an atmosphere having minimal oxygen, and may include the use of an antioxidant.
The structures of various natural and synthetic flavonoid compounds are known. For example, Heterocycles, Vol. 34, no. 6, pages 1213-1225 (Fukai, et al.) discloses the use of 1H-NMR in order to identify isoprenoid substituted flavanols. The structures of various flavonoids are also proposed.
Tetrahedron, Vol. 58, no. 18, pages 2619-2622 (Sutthivaiyakit, et al.) discloses the purification of various flavonoids from the chloroform extract of dried leaves of M. denticulata. 
The potential therapeutic use of certain flavonoid compounds as antioxidants for the treatment of patients having a disease or disorder involving oxidative damage, such as cancer, heart disease, neurological disorders, auto-immune disorders, ischemia-reperfusion injury, diabetic complications, septic shock, hepatitis, atherosclerosis and complications arising from HIV or Hepatitis B is known from. WO 2004/007475. WO 2004/007475 also discloses the potential application of the flavonoid compounds in sunscreen compositions and skincare compositions. In addition, WO 2004/007475 discloses the potential use of the flavonoid compounds as foodstuff stabilizers, where the ability of the compounds to combat free radicals is considered to be of utility in preventing or delaying the deterioration in food quality during storage. However, whilst WO 2004/007475 discloses the use of the flavonoid compounds for the in vivo therapeutic treatment of living matter and for the in vitro stabilizing treatment of dead matter, there is no disclosure or suggestion that the flavonoid compounds would be useful for in vitro preservation of living matter.
J. Agric. Food Chem., Vol. 48, pages 3876-3884 (Miranda et al.), disclosed test of certain flavanoid compounds to inhibit in vitro oxidation of human low density lipoprotein. British Journal of Cancer, 2003, Vol. 89(2), pages 357-362, and Vol. 89(11), pages 2140-2146, disclose the use of monoHER as providing protection against doxorubicin-induced inflammatory effects in vitro and against doxorubicin-induced cardiotoxicity in vitro, based on protection of human umbilical cord vascular endothelial cells and neonatal rat cardiac mycocytes, respectively. Both cell types are fully differentiated. Free Radical Biology & Medicine, 2002, Vol. 32(7), pages 596-604, disclose the testing of certain flavonoids for protecting primary cultures of rat cortical cells against oxidative stress. The cell types are fully differentiated. Bio. & Pharm. Bull., 2001, Vol. 24(12) pages 1373-1379, disclose in vitro protective effect of mixtures of extracts from certain plants, including some flavonoids, against oxidative stress on human skin cells. The cell types are fully differentiated. Bioorganic & Medicinal Chemistry, Vol. 12, no. 9, pages 2079-2098 (Bennett, et al.) discloses potentially therapeutic antioxidants wherein C6-C12 alkyl chains are attached to the A-ring of either a 3,3′,4′,5′-tetrahydroxyflavone or a 3,2′,4′,5′-tetrahydroxyflavone head group. J. Agric. Food Chem., Vol. 52, no. 6, pages 1514-1519 (Kajiya, et al.) disclosed tests of the antibacterial activity of certain flavonoid compounds. JP 2002 255810 A discloses catechin derivatives which possess improved antimicrobial properties in comparison to naturally occurring catechins. Journal of Pharmacy and Pharmacology, Vol. 55, no. 1, pages 131-142 (Kessler, et al.) and J. Chem. Soc. Vol. 6, pages 1215-1222 (Dangles, et al.) both disclose the biochemical evaluation of the effect of quercertin and quercertin derivatives such as its 3-O-glycoside rutin on lipid peroxidation. Both Kessler, et al. and Dangles, et al. suggest that flavonoids can act both as antioxidants and pro-oxidants under certain conditions.
WO 2008/062184, published after the priority date of the present application, discloses various flavonoid-type compounds which function as fluorescent probes and antioxidants and may be useful in discriminating healthy and stressed cells.
The object of the present invention is to provide an improved method for the in vitro preservation of living animal cells. In particular, it is an object of the present invention to provide an improved method for the in vitro preservation of a mammalian cell, tissue or organ for research or medical purposes e.g. transplantation. More preferred, it is an object of the present invention to provide an improved method for the in vitro preservation of stem cells.